The present invention relates to the field of protein engineering wherein changing amino acid compositions effects improvements in the nutrition content of feed and food. Specifically, the present invention relates to methods of enhancing the nutritional content of animal feed by expressing derivatives of a protease inhibitor to provide higher percentages of essential amino acids in plants.
Feed formulations are required to provide animals essential nutrients critical to growth. However, crop plants are generally rendered food sources of poor nutritional quality because they contain low proportions of several amino acids which are essential for, but cannot be synthesized by, monogastric animals.
For many years researchers have attempted to improve the balance of essential amino acids in the seed proteins of important crops through breeding programs. As more becomes known about seed storage proteins and the expression of the genes which encode these proteins, and as transformation systems are developed for a greater variety of plants, molecular approaches for improving the nutritional quality of seed proteins can provide alternatives to the more conventional approaches. Thus, specific amino acid levels can be enhanced in a given crop via biotechnology.
One alternative method is to express a heterologous protein of favorable amino acid composition at levels sufficient to obviate feed supplementation. For example, a number of seed proteins rich in sulfur amino acids have been identified. A key to good expression of such proteins involves efficient expression cassettes with tissue-preferred promoters. Not only must the gene-controlling regions direct the synthesis of high levels of mRNA, the mRNA must be translated into a stable protein and over-expression of this protein must not be detrimental to plant or animal health.
Among the essential amino acids needed for animal nutrition, often limiting in crop plants, are methionine, threonine, lysine, isoleucine, leucine, valine, tryptophan, phenylalanine, and histidine. Attempts to increase the levels of these free amino acids by breeding, mutant selection and/or changing the composition of the storage proteins accumulated in crop plants has met with limited success.
A transgenic example is the phaseolin-promoted Brazil nut 2S expression cassette. However, even though Brazil nut protein increases the amount of total methionine and bound methionine, thereby improving nutritional value, there appeared to be a threshold limitation as to the total amount of methionine that is accumulated in the seeds. The seeds remain insufficient as sources of methionine and methionine supplementation is required in diets utilizing soybeans.
An alternative to the enhancement of specific amino acid levels by altering the levels of proteins containing the desired amino acid is modification of amino acid biosynthesis. Recombinant DNA and gene transfer technologies have been applied to alter enzyme activity catalyzing key steps in the amino acid biosynthetic pathway. See Glassman, U.S. Pat. No. 5,258,300; Galili, et al., European Patent Application No. 485970; (1992); incorporated herein in its entirety. However, modification of the amino acid levels in seeds is not always correlated with changes in the level of proteins that incorporate those amino acids. See Burrow, et al., Mol. Gen. Genet.; Vol. 241; pp. 431-439; (1993); incorporated herein in its entirety by reference. Increases in free lysine levels in leaves and seeds have been obtained by selection for DHDPS mutants or by expressing the E. coli DHDPS in plants. However, since the level of free amino acids in seeds, in general, is only a minor fraction of the total amino acid content, these increases have been insufficient to significantly increase the total amino acid content of seed.
The lysC gene is a mutant bacterial aspartate kinase which is desensitized to feedback inhibition by lysine and threonine. Expression of this gene results in an increase in the level of lysine and threonine biosynthesis. However, expression of this gene with seed-specific expression cassettes has resulted in only a 6-7% increase in the level of total threonine or lysine in the seed. See Karchi, et al., The Plant J.; Vol. 3; pp. 721-7; (1993); incorporated herein in its entirety by reference. Thus, there is minimal impact on the nutritional value of seeds, and supplementation with essential amino acids is still required.
In another study (Falco et al., Biotechnology 13:577-582, 1995), manipulation of bacterial DHDPs and aspartate kinase did result in useful increases in free lysine and total seed lysine. However, abnormal accumulation of lysine catabolites was also observed suggesting that the free lysine pool was subject to catabolism.
Based on the foregoing, there exists a need for methods of increasing the levels of essential amino acids in seeds of plants. Previous approaches have led to insufficient increases in the levels of both free and bound amino acids and insignificant enhancement of the nutritional content of the feed.
It is the object of the present invention to provide nucleic acids and polypeptides relating to the enhancement of essential amino acids in plants.
It is another object of the present invention to provide antigenic fragments of the polypeptides of the present invention.
It is another object of the present invention to provide transgenic plants comprising the nucleic acids of the present invention.
It is another object of the present invention to provide methods making and expressing, in a transgenic plant, of the nucleic acids of the present invention.
It is another object that expression of the nucleic acids encoding the proteins of the present invention can be increased relative to a non-transformed control plant.
It is an object to provide a digestible substituted protein.
It is an object to provide a proteotypically stable, substituted protein, able to accumulate to useful levels in plants.
It is an object of this invention to provide a polypeptide with a non-native residue in more than about 11% to less than about 75% of the amino acid residues.
It is therefore an object of the present invention to provide methods for increasing the levels of one or more of a combination of essential amino acid in the seeds of plants used for animal feed.
It is a further object of the present invention to provide seeds for food and/or feed with higher levels of essential amino acid, than wild type species of the same seeds.
It is a further object of the present invention to provide seeds for food and/or feed such that the level of one or more of the essential amino acids is increased such that the need for feed supplementation is greatly reduced or obviated.
It is an object of the present invention to provide a Cl-2-like polypeptide with an increased level of essential amino acids through substitution of seven or more of the amino acid residues in a Cl-2-like polypeptide. Seven or more of positions 1, 8, 11, 17, 18, 19, 20, 22, 23, 31, 34, 38, 40, 41, 47, 49, 56, 58, 59, 60, 61, 62, 63, 65, 67, 69, 73, 75, 76, 78, 79, 81, 82, or combinations thereof.
Of the wild type protein are substituted with essential amino acid.
It is an object of the present invention to provide expression of the present chymotrypsin inhibitor derivatives in plants to provide higher percentages of essential amino acid in plants than wild type plants.
It is an object of this invention to provide a Cl-2-like polypeptide with increased stability.
It is an object of the present invention to provide methods for increasing the essential amino acid content of plants.
It is an object of the present invention to provide methods for increasing the nutritional value of a protein by altering a Cl-2-like polypeptide to enhance its nutritional value by substituting essential amino acids at positions corresponding to 1, 8, 11, 17, 18, 19, 20, 22, 23, 31, 34, 38, 40, 41, 47, 49, 56, 58, 59, 60, 61, 62, 63, 65, 67, 69, 73, 75, 76, 78, 79, 81, 82, or combinations thereof.
Choices of substitutions described herewithin are optionally grouped within parentheses and are separated by a semicolon. The native amino acid preceeds the position number using SEQ ID NO. 2 as a reference. The possible substitutions follow the residue number.